Richard Kolodner, PhD of University Of California, San Diego
Michael R. Stratton, FRCPath of Sanger Institute
Cancer is a genetic disease. Increased cancer susceptibility can result from inherited mutations in tumor suppressor genes and oncogenes as well other genes. In addition, in both inherited and the much more common sporadic cancers, mutations arise that drive the development and progression of the cancer. Decades of research have led to an understanding of the types of pathways that become genetically and epigenetically compromised in cancer; however, a comprehensive understanding of the genetics of all tumor types is not yet available. In recent years it has been possible to bring to the clinic therapies that are targeted to genetic defects found in cancer. The classic example is the treatment of Chronic Myelogenous Leukemia (CML). This disease is characterized by a specific chromosomal translocation resulting in the breakage and fusion of two genes to create the novel, mutant BCR-ABL fusion gene. The expression of this fusion gene drives tumorigenesis in this disease.
The development of a class of agents that inhibit the BRC-ABL oncoprotein, which is only expressed in CML cells, has dramatically improved the prognosis of patients diagnosed with CML. Many other such anti-cancer drugs have been developed or are under development for treating other types of cancer. However, the ability to more broadly implement these types of therapies will require a much more comprehensive understanding of the genetic and epigenetic changes in different cancers. Since the successful sequencing of the human genome, there have been rapid advances in the development of genomic technologies.
These new technologies, which are advancing and evolving at a rapid rate, have made it possible to gain a much more detailed understanding of the types of genetic changes present in cancers. For example, it is now possible to detect genetic changes that occur at low frequencies in different cancer types and begin to explore whether such mutations are bystander mutations or whether they influence the development and progression of cancer, and therefore might guide the development of targeted therapeutics. In addition, it is also possibly to map the types of genome rearrangements that occur in cancers in much greater detail.
In parallel with these developments has been the development of sophisticated bioinformatics needed for analyzing the much larger data sets being generated. Over the next few years, it is likely that our ability to explore the genetics of individual cancers will expand by a previously unanticipated extent. In this rapidly evolving field, the meeting, to be led by Dr. Richard Kolodner of the Ludwig Institute for Cancer Research and Dr. Michael Stratton of the Sanger Center, will provide an important opportunity to explore our current state of knowledge of Cancer Genomics and the directions in which the field is moving.
The focus of the 2010 Forbeck Foundation was on the rapidly moving field of cancer genomics. It is generally accepted that cancer is caused by the accumulation of mutations or epigenetic modification that affect specific genes. The identity of the affected genes provides clues to the cellular processes underlying tumorigenesis and has proven useful for diagnostic and therapeutic purposes. To date, however, only a small fraction of the genes has been analyzed and the number and type of alterations responsible for the development of common tumor types are not well known. Much of our current knowledge of the genetic and epigenetic alterations in cancers has come from decades of work cloning and analyzing individual genes in human cancers. The insights from these studies have provided diagnostic tools for analyzing inherited cancer susceptibility and linking genetic changes in cancer to sensitivity and resistance to chemotherapy as well as providing predictions of prognosis and survival.
A key insight in the applicability of cancer genetics to cancer treatment came from the development of drugs that successfully target the oncoprotein encoded by the BCR-ABL translocation, a genetic alteration that underlies Chronic Myelogenous Leukemia. This development has led to the common view that many other successful cancer treatments that target genetic alterations, so-called personalized therapy, can be developed. However, implementation of personalized therapies will require a much more comprehensive knowledge of the genetic and epigenetic changes in different cancers.
The completion of the human genome sequence greatly facilitated the types of studies required to identify the diversity of genetic and epigenetic changes in different cancers. In the last several years, facilitated by the knowledge of the human genome sequence, there has been the rapid development of ever more powerful methods for analyzing genetic and epigenetic changes on a genome-wide scale. The rapidly decreasing cost of genome-wide analysis and the continued development of bioinformatics methods have brought us to the point where the results of genome-wide analysis of genetic and epigenetic changes are becoming available for many different tumor types.
The 2010 Conference brought together a group of experts in Cancer Genomics to discuss progress and the challenges in transforming the results of current Cancer Genomics initiatives into practical treatments that can impact the prognosis for cancer patients. A session chaired by Dr. Joe Gray focused on the genomic analysis of a number of cancer genomes, with a primary focus on the use of whole genome and whole exome sequencing methods. The discussions in this session also triggered a debate about how to distinguish which identified mutations were “driving” cancer from those that are merely “passengers” and whether or not this driver/passenger dichotomy was even useful or legitimate for understanding cancer biology.
Dr. James Downing discussed collaborative efforts to perform large-scale genomic analysis of leukemias using sequencing approaches to detect mutations and genome rearrangements and array based methods to detect copy number changes. These efforts were directed at both understanding the genetic changes in primary leukemias as well as in understanding relapse cases. These efforts have revealed information about the prevalence of mutations in different cancer-relevant genes, such as PAX5, in different leukemias as well as demonstrating how copy number changes can lead to clinically relevant alterations in gene expression. Mutations that can predict relapse have been identified, and interestingly, it was demonstrated that relapses could result from leukemia cells present in the original cancer population but are distinct from the major clone present in the original leukemia. Finally, recent results on mutations affecting DNA methyl transferase genes in leukemias were discussed providing an interesting linkage between the development of some leukemias and epigenetic processes.
Dr. David Huntsman discussed his efforts to use whole-genome analysis to address clinical questions in ovarian cancer. Of particular importance in this disease is the development of molecular markers for sub-classifying ovarian cancer to better understand both prognosis and treatment options. Ovarian cancers with BRCA1 or BRCA2 mutations are sensitive to PARP inhibitors, and these mutations are currently used to select patients for PARP inhibitor therapy. Conversely, he showed that there are PARP inhibitor sensitive ovarian cancers without defects in BRCA1 or BRCA2, illustrating the need for a molecular signature for identifying this ovarian cancer sub-type. Related to this problem, Huntsman also described ongoing genomic analysis of ovarian cancers that had acquired resistance to PARP inhibitors to understand the mechanism of resistance.
Dr. Victor Velculescu discussed his laboratory’s collaborative efforts to systematically study the cancer genome through analysis of the majority of protein coding genes in breast, colorectal, pancreatic, ovarian and brain cancers. Their analysis to date has focused on direct resequencing of all of the currently annotated genes in the human genome. They have identified mutations both in genes previously known to be mutated in human cancer and in a wealth of new mutated genes, implicating new pathways as playing roles in human cancer. Their results indicate that the genetic landscape of human cancers include both commonly mutated genes as well as genes that are mutated at lower frequencies. Importantly, their data has allowed them to calculate the number of driver mutations in different cancer types and show that each type of cancer has a different, but characteristic, number of driver mutations. These studies define the genetic landscape of human cancers, open fertile avenues for basic research in tumor biology, and provide new targets for personalized diagnostic and therapeutic intervention.
Dr. Tom Hudson discussed examples of ongoing initiatives that were established to accelerate the translation of new cancer mutation discoveries into clinical applications. He focused on inherited common risk alleles that were identified by Genome-Wide Association Studies (GWAS). Individually, these common alleles confer only small increases in risk of developing colorectal cancer (CRC), but by combining these alleles with age and family history, they observed that population subgroups can be identified with a predicted absolute CRC risk sufficiently high as to merit surveillance/intervention. He also focused on the analysis of somatic mutations acquired during the development of tumors. Here, the International Cancer Genome Consortium (ICGC) was formed with the aim of generating comprehensive catalogues of genomic abnormalities (somatic mutations, abnormal expression of genes, epigenetic modifications) in tumors in 50 different cancer types and/or subtypes and to make the data available to the research community. In this context, Hudson presented a design for a clinical trial underway in Canada that is sequencing a large set of cancer genes in tumors from patients with advanced cancer, in order to select therapies that are based on mutation status.
A session chaired by Dr. Victor Velculescu focused on the structure of cancer genomes integrating a number of approaches including analysis of copy number variation, mapping of genome rearrangements and both whole genome and targeted gene sequencing. Dr. Sharon Diskin (Forbeck Scholar) discussed her laboratory’s efforts to understand the genetic and environmental factors that cause sporadic neuroblastoma (NB), which remain largely unknown. They have been comparing germline genome-wide single nucleotide polymorphism (SNP) genotypes from 5,000 NB patients to 10,000 controls in order to discover SNP and copy number variation (CNV) associations. To date, they have genotyped over 3,800 neuroblastoma cases and have reported six loci containing common SNPs and a common CNV each highly associated with sporadic NB. To begin to understand the biological relevance of these associations, they are performing correlative and mechanistic studies in lymphoblastoid cell lines (LCLs) as well as matched primary NB tumor tissues and NB cell line models. Together, their data provide evidence for at least two distinct genetic subtypes of NB at the level of tumor initiation, and illustrate the utility of combining germline and somatic data in assessing GWAS signals in cancer. Additional NB associated variants, both common and rare, have been identified and validation efforts are ongoing.
Dr. Michael Stratton primarily focused on his laboratory’s use of Next Generation DNA sequencing technologies to perform high-density mapping and sequencing of the genome rearrangements found in human tumors. Their data indicated that each tumor had its own genome rearrangement signature. A particularly interesting use of this information was illustrated by a study of several patients in which it was possible to follow the genome rearrangement signatures for the primary tumor and subsequent relapse and metastatic tumors. This allowed them to characterize tumor clonality and evolution through the course of disease development and progression.
Dr. Rick Wilson described the development and use of Next Generation sequencing technology at the Sequencing Center at Washington University in St. Louis to study cancer genomes. The work he described illustrated that early stage sequencing can sometimes miss important mutations and that there is considerable value to continuing to analyze larger numbers of tumors with newer, improved technology as this can reveal important cancer-related genetic variation. The work also illustrated the enormous amount of genetic variation in tumors that is being revealed by sequencing studies and the enormous challenge this presents in classifying these potential genetic changes, validating the genetic changes and ultimately determining their significance to cancer biology.
Dr. Christopher Putnam (Forbeck Scholar) discussed his efforts to understand the nature of genome instability. The genomes of many types of cancers are extensively rearranged and show evidence of ongoing instability. This instability is likely to be due to accumulated defects in processes that maintain normal genomes in non-cancerous cells and such defects could play a role in initiation or progression of cancer. He described studies of genome instability in a model organism, the yeast Saccharomyces cerevisiae. These studies have identified genes in which mutations result in increased rates of accumulating genome rearrangements and have revealed that the types of genome rearrangements that occur, which resemble those discovered in human tumors, are strongly influenced by which underlying genetic defects causes the increased genome instability observed. These studies also suggest that genetic defects underlying increased genome instability might be identified through analysis of the spectrum of genome rearrangements present in an individual tumor and, because many of these genetic defects give rise to sensitivity to DNA damaging agents, that rearrangement spectra could eventually be used to help guide treatment choice.
Dr. Jessica Zucman-Rossi discussed her team’s efforts to identify new carcinogenesis pathways and provide molecular classifications for benign and malignant liver tumors using functional genomics approaches. Their strategy enabled them to identify a broad diversity of genetic lesions that will have important implications for patients with liver disease. For example, dissection of molecular pathways in hepatocellular adenoma identified new risk factors (obesity) and genetic susceptibility (germline HNF1A and CYP1B1 mutations) that promote the development of benign liver tumors. Moreover, new pathways of tumorigenesis (HNF1A, STAT3, ß-catenin and others) showed a close relationship with the clinical and pathological features of both tumors and patients. As a result of this analysis, it was possible to identify small homogeneous sub-groups of tumors which will allow modification of prevention, diagnosis and treatment strategies for patients leading to more efficient personalized treatment.
A session chaired by Dr. Tom Hudson, focused on evaluating the significance of genetic variation in cancer genomes through the identification genetic and epigenetic changes that are of significance to the development of cancer. Dr. David Adam’s work is focused on using functional assays in mice as a model system to identify tumor suppressor genes. He described the largest and most comprehensive forward genetic screen in the mouse for genes that play a role in tumorigenesis. The screen was performed in Apc mutant mice for bowel cancer genes. His critical finding was that there were many more genes than previously thought that can contribute to tumor formation. These results challenge the dogma that that there are just a few major genes in which defects result in cancer formation and suggested that as many as 20 genes can contribute to tumorigenesis in each clonal cancer. Interestingly, this number of genes falls within the range of the number of driver mutations in individual cancer identified by Velculescu’s studies.
Dr. Andrea Califano discussed the use of Systems Biology to identify critical pathways in different cancers. Reverse engineering of cell context specific molecular interactions has unveiled the complex structure and control of regulatory networks in normal cell physiology and in disease. Interrogation of these networks using cancer specific signatures has identified an intermediate layer of transcriptional regulation, responsible for the integration of a broad spectrum of signals that are altered in specific tumor subtypes. For instance, his laboratory demonstrated that a small module regulated by the two transcription factors C/EBP(b/d) and Stat3 is responsible for the synergistic integration of genetic alterations contributing to expression of mesenchymal genes in High Grade Glioma. Activation of the mesenchymal program is associated with the poorest prognosis in these tumors. Similarly, Nf-kB was shown to integrate aberrant signals associated with an entire spectrum of genetic alterations in upstream signal transduction genes, such as CARD11, A20, TRAF2, TRAF5, TAK1, and RANK, among others, specific to the more aggressive ABC subtype of Diffuse Large B Cell Lymphoma compared to the GCB subtype. These results suggest that while sequencing can identify key oncogenes and tumor suppressors associated with tumor initiation and tumor progression, analysis of regulatory networks can provide a complementary and valuable perspective by identifying genes acting as key integrators of these aberrant signals. These approaches have the potential to provide more general targets for single- and combination drug therapy as well as more informative and universal biomarkers that are causally relate to the presentation of the tumor subtype.
Dr. Richard Kolodner discussed his laboratory’s efforts to use the yeast Saccharomyces cerevisiae as a model system to identify the genes and pathways that cells use to prevent genome instability. It is well known that many cancers have ongoing genome instability that potentially reflects a defect in a gene or pathway required for maintenance of genome stability, and work with PARP inhibitors has suggested that such defects may be a cancer therapeutics target. However, the range of genetic defects that result in increased genome instability is largely unknown. A series of quantitative genetic assays for detecting increased genome instability has been developed and through a combination of genetic screens and bioinformatics analysis, a series of 510 yeast genes predicted to act to prevent genome instability has been developed and partially validated. Initial attempts to mine cancer genome mutation data sets to determine if the human homologues of these genes are mutated in human cancers has suggested that a significant number of these genes are potentially mutated in some human cancers; however, functional studies will be required to validate the presumptive mutations.
Dr. Peter Laird discussed his work demonstrating that genomic loci that are involved in cellular differentiation and are targeted by Polycomb Group Repressors in embryonic stem cells are predisposed to become methylated in cancer cells, suggesting that an epigenetic block to cellular differentiation may sometimes be an initiating event in carcinogenesis. DNA methylation changes contribute directly to cancer by transcriptional silencing of tumor-suppressor genes through promoter CpG island hypermethylation. His group demonstrated very strong associations between distinct epigenetic subtypes, such as CpG Island Methylator Phenotypes (CIMP) and specific somatic genetic events, such as BRAF mutation in colorectal cancer and IDH1 mutation in glioblastoma multiforme. These associations are consistent with an early role for DNA methylation, and suggest these epigenetic modifications provide a cellular context for the subsequent somatic mutation that is favorable for disease progression. This session, chaired by Dr. Jessica Zucman-Rossi, focused on the prospects and challenges for using the results of genomic analysis to direct cancer therapy.
Dr. Benjamin Berman (Forbeck Scholar) discussed his efforts to develop DNA methylation profiles for evaluating different cancers. Epigenetic mechanisms play important roles in the development of cancer, and DNA methylation has been used as both a biomarker and a target for therapeutics. He applied a new sequencing technology (massively parallel MethylCseq) to a paired colon tumor and normal colon mucosa sample to determine the complete map of DNA methylation at single base-pair resolution, the first such "methylome" map of a tumor. This high resolution map revealed a number of novel features, including the presence of methylation differences at two distinct spatial scales. Long (>100 kb) blocks of reduced methylation in the tumor corresponded to silencing domains associated with the nuclear periphery, and within these were thousands of short (~1 kb) CpG island elements with increased methylation in the tumor. Profiling additional types of cancer in this way is expected to yield insights into the regulatory changes that occur in the development of cancer, and guide the use of different treatment regimens.
Dr. Levi Garraway discussed the many challenges involved in bringing genomic information into clinical practice. The key questions he discussed are whether ongoing genomic studies will provided all of the clinically relevant data needed to drive future application, what are the challenges in actually using genomic information to guide clinical decisions, how do we use genomic information in the clinical setting so that we know it is actually useful and what types of clinical and translational research will be needed once genomic information is readily available. Appropriately addressing these questions will be required to ensure that patients fully benefit from cancer genomics.
Dr. Derek Chiang (Forbeck Scholar) discussed his studies aimed at understanding the many mechanisms of drug resistance in tumors and the identification of therapeutic targets that will be suitable for developing combination therapies that can target drug resistant tumors. Toward this end, they have performed an RNA interference screen to identify kinases that may confer sensitivity to sorafenib combinations. Computational analyses of signaling pathways identified in this screen found a surprising but potentially useful link between Toll-like receptor signaling and sorafenib in liver cancer cell lines.
Dr. Joe Gray focused on the use of “omic” information on breast cancer to address the poor survival duration (<25% 5 year survival) experienced by patients with metastatic breast cancer. One approach he suggested for dealing with this problem was early detection of metastasis prone breast cancer. He described their efforts to develop sensitive and specific detection reagents for MRI/PET imaging and multiplex immunohistochemical detection designed for specific detection of cancers that will metastasize with high probability. These studies are being guided by “omic” information on gene expression, alternative mRNA splicing, fusion genes, DNA methylation profiles, copy number abnormalities and promoter methylation. The second approach he suggested for dealing with this problem was improved treatment of metastatic disease. He described their efforts to screen next generation drugs singly and in combination in a panel of well-characterized cell lines where complete genomic profiles are becoming available to identify molecular determinants of response. They are focusing on identification of response predictors that are not influenced by variations in the microenvironment, in order to make therapies robust across the diverse microenvironments in which metastatic tumor cells reside.
In summary, the 2010 conference was an exciting meeting held at a pivotal time in the development of the field of Cancer Genomics. The discussion revealed that the wealth of genomic information about cancer that is currently being generated suggests that we are on the verge of having enough data to make personalized cancer treatment a reality for many different types of cancer. Yet, as exciting as the promises of these new advances are, the discussion also revealed that key challenges remain in the interpretation and integration of this data in ways that will allow these results to be applied routinely in the clinic.
Quotes from Participants
“The meeting last weekend was a clear example of how your group has had a direct impact on the enterprise of cancer research. The meeting was unlike any other I have attended. It was fantastic to be able to get together with the leaders of the field of cancer genomics and discuss in an open forum where the field has been, and where it needs to go. The combination of scientific sessions, breaks, lunch out on the point and the leisurely walks back to the hotel, and the evening receptions provided the ideal balance of setting to allow the participants to not only discuss science, but to also interact outside the bounds of our often competitive existence. Again, thank you for everything you do.” -- James R. Downing, M.D., St. Jude Children's Research Hospital, Memphis, TN
“The meeting really was special and unlike any other I've attended before. Having just 12 participants and restricting the number of slides really helps promote discussion.” -- David Adams, Experimental Cancer Genetics, Wellcome Trust Sanger Institute, Cambridge, UK
David Adams, PhD
Wellcome Sanger Institute
Benjamin P. Berman, PhD
University of Southern California
Andrea Califano, PhD
Derek Y. Chiang, PhD
"Novartis Institutes for BioMedical Research, Inc."
Sharon J. Diskin, PhD
Children's Hospital of Philadelphia
James R. Downing, MD
St. Jude Children's Research Hospital
Levi Garraway, MD, PhD
Dana-Farber Cancer Institute
Joe W. Gray, PhD
Lawrence Berkeley National Laboratory
Tom Hudson, MD
Ontario Institute for Cancer Research
David Huntsman, MD
University of British Columbia
Richard Kolodner, PhD
University of California San Diego
Peter W. Laird, PhD
University of California Los Angeles
Chris Putnam, PhD
Ludwig Institute for Cancer Research
Wellcome Sanger Institute
Victor Velculescu, MD, PhD
Johns Hopkins Medicine
Richard K. Wilson, PhD
Washington University in Saint Louis
Jessica Zucman-Rossi, MD, PhD